- Many of our deepest insights into the biology of aging have been gained using the simple Eukaryote Saccharomyces cerevisiae. Foundational studies using yeast have led to the discovery of the role played by the silent information regulator protein Sir2 in modulating replicative lifespan, and the roles played by Ras2, Cyr1 and Sch9 in modulating chronological lifespan. Each of these genes has a homologue with similar function in higher Eukaryotes, including mammals. These discoveries have generated excitement about the prospects for extending lifespan and improving quality of life for older members of the human population. To date, all investigations into the genetic mechanisms that extend chronological lifespan in yeast have focused on the viability of non-growing planktonic cells in stationary-phase batch culture. There, cells enter a physiological state where metabolism is reprogrammed to efficiently use stored resources for the purpose of cell maintenance. Not surprisingly, screens for mutants which alter chronological lifespan under these conditions have produced a collection of genes whose activities influence resistance to stress. But yeast populations largely cease to reproduce in another, very different environmental context. Continuously or semi-continuously fed bioreactors populated by yeast immobilized in semi-solid beads can be maintained for weeks, or even months. These reactors continue to produce close-to-theoretical yields of ethanol but very little biomass, relative to substrate input. Herein, we present demographic, physiological and genomic data that illuminate features of the immobilized state, features that we contend make this system ideally suited for the study of chronological aging. We propose to follow on from these results with an investigation into the mechanism(s) that enable yeast to live for extended periods of time in a calorically un-restricted environment wherein metabolic flux appears to be maximized at the expense of cell growth and reproduction. The Specific Aims of this investigation are threefold;we will: (1) Determine in wild-type yeast whether long-term immobilization alters chronological lifespan and global gene expression relative to aging planktonic cultures;(2) Establish, relative to wild-type, whether mutations known to alter both replicative and chronological lifespan in planktonic cells produce similar alterations in aging immobilized cells. (3) Conduct a large-scale genetic screen of single-gene-deletion yeast strains cultured in the immobilized and planktonic states (a) to discover new classes of longevity genes that act in undescribed pathways, and (b) to establish whether there are common determinants of chronological longevity under calorically-restricted and non-calorically-restricted conditions. PROJECT NARRATIVE - Many of our deepest insights into the biology of aging have been gained using the simple microbe, Bakers yeast. Individual yeast cells have a finite lifespan, and they age in two ways: mother cells age as they undergo successive rounds of cell division;non-dividing cells also age and eventually die. Caloric restriction appears to extend lifespan in all species studied, including yeast. But controversy surrounds the issue of whether caloric restriction is a proximal or ultimate cause of longevity. To date, all studies on the genetics on non-dividing cells have imposed caloric restriction, making this variable impossible to tease out. Yeast can be confined within semi-solid alginate beads and exposed to excess nutrients. There, they convert sugar to ethanol at extremely high rates, but do so with little or no cell division. We will exploit this system as a tool to discover whether lifespan extension is possible under calorically non- restrictive conditions, and if so, we aim to discover underlying genetic determinants.